This invention relates to a mammalian cDNA which encodes a mammalian Ras association domain containing protein and to the use of the cDNA and the encoded protein in the diagnosis and treatment of cell proliferative and inflammatory disorders.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural is features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Signal transduction is the general process by which cells respond to extracellular signals. In typical signal transduction pathways, binding of a signaling molecule such as a hormone, neurotransmitter, or growth factor to a cell membrane receptor is coupled to the action of an intracellular second messenger. G protein-coupled receptors (GPCRs) control intracellular processes through the activation of guanine nucleotide-binding proteins (G proteins). G proteins are heterotrimeric and consist of xcex1, xcex2, and xcex3 subunits. The xcex1 subunit contains a guanine nucleotide binding domain and has GTPase activity. When GTP binds to the xcex1 subunit, it dissociates from the xcex2 and xcex3 subunits and interacts with cellular target molecules. Hydrolysis of GTP to GDP serves as a molecular switch controlling the interactions of the xcex1 subunit with other proteins. The GDP bound form of the xcex1 subunit dissociates from its cellular target and reassociates with the xcex2 and xcex3 subunits. A number of accessory proteins modulate G protein function by controlling their nucleotide state or membrane association. These regulatory molecules include exchange factors (GEFs) which stimulate GDP-GTP exchange, GTPase activating proteins (GAPs) which promote GTP hydrolysis, and guanine nucleotide dissociation inhibitors (GDIs) which inhibit guanine nucleotide dissociation and stabilize the GDP-bound form. G proteins can be classified into at least five subfamilies: Ras, Rho, Ran, Rab, and ADP-ribosylation factor, and they regulate various cell functions including cell growth and differentiation, cytoskeletal organization, and intracellular vesicle transport and secretion.
The Ras subfamily transduces signals from tyrosine kinase receptors, non-tyrosine kinase receptors, and heterotrimeric GPCRs (Fantl et al. (1993) Annu Rev Biochem 62:453-481; Woodrow et al. (1993) J Immunol 150:3853-3861; and Van Corven et al. (1993) Proc Natl Acad Sci 90:1257-1261). Stimulation of cell surface receptors activates Ras which, in turn, activates cytoplasmic kinases that control cell growth and differentiation. The first Ras targets identified were the Raf kinases (Avruch et al. (1994) Trends Biochem Sci 19:279-283). Interaction of Ras and Raf leads to activation of the MAP kinase cascade of serine/threonine kinases, which activate key transcription factors that control gene expression and protein synthesis (Barbacid (1987) Ann Rev Biochem 56:779-827; Treisman (1994) Curr Opin Genet Dev 4:96-101). Mutant Ras proteins, which bind but do not hydrolyze GTP, are constitutively activated, and cause continuous cell proliferation and cancer (Bos (1989) Cancer Res 49:4682-4689; Grunicke and Maly (1993) 4:389-402).
Ras regulates other signaling pathways by direct interaction with different cellular targets (Katz and McCormick (1997) Curr Opin Genet Dev 7:75-79). One such target is Ral GDS, a guanine nucleotide dissociation stimulator for the Ras-like GTPase, Ral (Albright et al. (1993) EMBO J 12:339-347). Ral GDS couples the Ras and Ral signaling pathways. Epidermal growth factor (EGF) stimulates the association of Ral GDS with Ras in mammalian cells, which activates the GEF activity of Ral GDS (Kikuchi and Williams (1996) J Biol Chem 271:588-594; Urano et al. (1996) EMBO J 15:810-816). Ra1 activation by Ral GDS leads to activation of Src, a tyrosine kinase that phosphorylates other molecules including transcription factors and components of the actin cytoskeleton (Goi et al. (2000) EMBO J 19:623-630). Ral interacts with a number of signaling molecules including Ra1-binding protein, a GAP for the Rho-like GTPases; Cdc42 and Rac, which regulate cytoskeletal rearrangement; and phospholipase D1, which is involved in vesicular trafficking (Feig et al. (1996) Trends Biochem Sci 21:438-441; Voss et al. (1999) J Biol Chem 274:34691-34698).
Norel was identified from a yeast two-hybrid screen as a protein that interacts with Ras and Ras-related protein, Rap1b (Vavvas et al. (1998) J Biol Chem 273:5439-5442). It is a highly basic protein (pI=9.4) of 413 amino acids that contains a cysteine-histidine-rich region predicted to be a diacylglycerol/phorbol ester binding site, a proline-rich region at its N-terminus that may be an SH3 binding domain, and a Ras/Rap binding domain located at its C-terminus. Nore1 binds Ras in vitro in a GTP dependent manner. Experiments in vivo show that the association of Nore1 with Ras is dependent on EGF and 12-O-tetradecanoylphorbol-13-acetate activation in COS-7 cells and on EGF in KB cells.
Ras and other G proteins play roles in regulating the immune inflammatory response. Granulocytes, which include basophils, eosinophils, and neutrophils, play critical roles in inflammation. Eosinophils release toxic granule proteins, which kill microorganisms, and secrete prostaglandins, leukotrienes and cytokines, which amplify the inflammatory response. They sustain inflammation in allergic reactions and their malfunction can cause asthma and other allergic diseases. Interleukin-5 is a cytokine that regulates the growth, activation, and survival of eosinophils. The signal transduction mechanism of IL-5 in eosinophils involves the Ras-MAP kinase and Jak-Stat pathways (Pazdrak et al. (1995) J Exp Med 181:1827-1834; Adachi and Alam (1998) Am J Physiol 275:C623-633). Raf-1 kinase activation by Ras is implicated in eosinophil degranulation.
Neutrophils migrate to inflammatory sites where they eliminate pathogens by phagocytosis and release toxic products from their granules that kill microorganisms. G proteins, including Ras, Ral, Rac 1 and Rap1 regulate neutrophil function (M""Rabet et al. (1999) J Biol Chem 274:21847-21852). Rac1 may be involved in the respiratory burst of neutrophils. Ras and Rap1 are activated in response to the chemotactic agent, formyl methionine leucine phenylalanine (fMLP); the lipid mediator, platelet activating factor (PAF); and the cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). Both Ras and Rap1 appear to play roles in neutrophil activation. Ral is activated by fMLP and PAF but not by GM-CSF and may be involved in chemotaxis, phagocytosis, or degranulation. Impairment of neutrophil function is associated with various inflammatory and autoimmune diseases.
The discovery of a mammalian cDNA encoding a new Ras target or effector satisfies a need in the art by providing compositions which are useful in the diagnosis and treatment of cell proliferative and inflammatory disorders.
The invention is based on the discovery of a mammalian cDNA which encodes a Ras association domain containing protein (RADCP) which is useful in the diagnosis and treatment of cell proliferative and inflammatory disorders, particularly thymus hyperplasia, allergies, asthma, and hypereosoinophilia.
The invention provides an isolated mammalian cDNA or a fragment thereof encoding a mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having 80% identity to the amino acid sequence of SEQ ID NO:1, an antigenic epitope of SEQ ID NO:1, an oligopeptide of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides an isolated mammalian cDNA or the complement thereof selected from the group consisting of a nucleic acid sequence of SEQ ID NO:2, a variant having at least 87% identity to the nucleic acid sequence of SEQ ID NO:2, a fragment of SEQ ID NOs:3-9, an oligonucleotide of SEQ ID NO:2. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding RADCP. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make RADCP. The invention still further provides a transgenic cell line or organism comprising the vector containing the cDNA encoding RADCP. The invention additionally provides a mammalian fragment or the complement thereof selected from the group consisting of SEQ ID NOs:10-12. In one aspect, the invention provides a substrate containing at least one of these fragments. In a second aspect, the invention provides a probe comprising the fragment which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single stranded complementary RNA or DNA molecule.
The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose cell proliferative and inflammatory disorders, particularly thymus hyperplasia, allergies, asthma, and hypereosinophilia. In another aspect, the cDNA or a fragment or a complement thereof may comprise an element on an array.
The invention additionally provides a method for using a cDNA or a fragment or a complement thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, enhancers, repressors, and regulatory molecules.
The invention provides a purified mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having 80% identity to the amino acid sequence of SEQ ID NO:1, an antigenic epitope of SEQ ID NO:1, an oligopeptide of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides a composition comprising the purified protein or a portion thereof in conjunction with a pharmaceutical carrier. The invention further provides a method of using the RADCP to treat a subject with a cell proliferative or inflammatory disorder comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand , the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with a cell proliferative or inflammatory disorder.
The invention provides a method of using a mammalian protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions that allow specific binding, dissociating the antibody from the bound-protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of cell proliferative and inflammatory disorders, particularly thymus hyperplasia, allergies, asthma, and hypereosoinophilia.
The invention also provides a method of using a mammalian protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified antibodies.
The invention provides a purified antibody which bind specifically to a polypeptide comprising the amino acid sequence selected from SEQ ID NO:1 and fragments thereof. The invention also provides a method of using an antibody to diagnose cell proliferative and inflammatory disorders, particularly thymus hyperplasia, allergies, asthma, and hypereosoinophilia comprising combining the antibody comparing the quantity of bound antibody to known standards, thereby establishing the presence of cell proliferative and inflammatory disorders. The invention further provides a method of using an antibody to treat cell proliferative and inflammatory disorders comprising administering to a patient in need of such treatment a pharmaceutical composition comprising the purified antibody.
The invention provides a method for inserting a marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:2-12, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.